Treatment of neurological conditions by an interleukin-1 inhibiting compound

ABSTRACT

The use of a compound which prevents, inhibits or modifies the action of interleukin-1 as an active agent for the treatment of conditions of neurological degeneration. The active agent may be IL-1 receptor antagonist, particularly recombinant IL-1 ra.

FIELD OF THE INVENTION

This invention relates to a method for treatment of neurologicalconditions and to compositions and products useful for such treatment.

BACKGROUND OF THE INVENTION

Neurological conditions pose serious clinical problems, as their effectsare severe and long-lasting but little is known of any really effectivemeans for curing or even controlling them.

There is, therefore, a considerable need for some treatment for suchconditions.

SUMMARY OF THE PREFERRED EMBODIMENTS

According to a first aspect of the present invention there is providedthe use of a compound which prevents, inhibits or modifies the action ofinterleukin-1 as an active agent for the treatment of conditions ofneurological degeneration.

According to a second aspect of the present invention there is provideda formulation adapted for the use as defined in the previous paragraph,comprising an active agent as defined therein dispersed or dissolved ina pharmaceutically acceptable carrier (solvent or diluent), especiallyin water or an aqueous medium, especially in normal saline (an isotonicsolution of sodium chloride in water).

According to a third aspect of the present invention there is provided amethod of treating neurological degenerative conditions in a human oranimal patient comprising administration as an active agent a compoundwhich prevents, inhibits or modifies the action of interleukin-1.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating preferredembodiments of the present invention, are given by way of illustrationand not limitation. Many changes and modifications within the scope ofthe present invention may be made without departing from the spiritthereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more readily understood by referring to theaccompanying drawings in which

FIGS. 1A-B illustrate the effect of IL-1 receptor antagonist protein(IL-1 ra) on neuronal damage after cerebral ischemia. (B) The lowerpanel shows the volume of damage (mm³, computed from the volume underthe curve for upper panel, A). Mean±SEM, one way ANOVA, *P<0.05, and

FIGS. 2A-B illustrate the effect of IL-1 receptor antagonist protein onNMDA receptor mediated neuronal damage. (A) Upper panel shows the areaof damage (mm²) and (B) the lower panel shows the lesion volume (mm³,computed from the volume under the curve for upper panel). Mean±SEM,unpaired Students t-test, *P<0.001.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Interleukin-1 is commonly referred to as "IL-1".

The active agent thus defined has the effect of protecting neurons fromadverse effects, i.e. neuro-degeneration.

The active agent used may be in a variety of forms, for example anaturally occurring product or one produced by artificial or syntheticmethods, for example a genetically engineered form. Particularlysuitable is interleukin-1 receptor antagonist protein (convenientlyreferred to as "IL-1ra"), particularly recombinant IL-1 ra. It is alsopossible to use analogues of IL-1 ra, as well as derivatives andfragments thereof (and analogues of these compounds).

Our treatment is useful for a variety of conditions of neurologicaldegeneration, however caused, though the means by which the active agentwe specify here works is not yet clearly understood. It is believed thatit is probably by blocking the action of interleukin-1. The invention isespecially applicable to treatment of the neurons in the brain,periphery and spinal chord.

Cerebral lesions in several chronic neurodegenerative conditions (e.g.Alzheimer's disease (AD) and Down's Syndrome) are known to be associatedwith the formation of beta-amyloid (beta-AP), apparently due to abnormalmetabolism of beta-amyloid precursor protein (beta-APP) with consequentdeposition of beta-AP.

IL-1 has been demonstrated to be present in the brains of patients withDown's Syndrome and Alzheimer's Disease (Griffin et al, 1989, Proc NatAcad Sci 86, 7611) and in separate studies has been shown to inducesynthesis of the precursor of beta-APP (Goldberg et al, 1989, Proc NatAcad Sci 86, 7606).

Furthermore chronic degenerative processes associated withover-expression of beta-APP for an extended period can lead to loss ofsynapses, deposition of beta-AP and degeneration of neurons. We haveshown that this process is related to emergence of the clinical symptomsof cognitive and neurological deficits and demention (Gentleman S. M. etal. (1991). Neuropath.Applied Neurobiol.17,531). Our experimental datashow that these mechanisms are probably active in many of the diseasesdescribed in Chapters 5 and 6 of the International Classification ofDisease (10th edition) (e.g. Dementia in Alzheimer's disease,Parkinson's Disease, Cortical Lewy Body Disease, etc.)

Interleukin-1 (IL-1), a 17 KDa cytokine is synthesised (W. L. Farrar etal., Immunol.Rev. 100, 361, 1981) and acts within the central nervoussystem to mediate several aspects of the acute phase response, anddirectly modifies neuronal and glial function. An endogenous IL-1receptor antagonist (IL-1ra) has been identified (S. P. Eisenberg etal., Nature, 343, 341, 1990) which binds to IL-1 receptors in peripheraltissues and hippocampal neurones, and has been shown to inhibit manyperipheral actions of IL-1.

Further evidence that IL-1ra may be of benefit derives from observationsthat the concentrations of IL-1 and related cytokines are increased inbrain in response to traumatic injury, cerebral ischaemia, and HIVinfection and administration of IL-1 worsens ischaemic brain damage.(Gentleman et al (1993) Prog. Exp. Brain Res.(in press)) Thus, blockingits actions by administration of IL-1ra or related inhibitors of IL-1action may limit many forms of neurological damage.

The molecular weight of the active agent defined herein may vary over aconsiderable range. It may thus usually be of molecular weight up toabout 40 KDa (kilodaltons)--though products of higher or lower molecularweight may be used if desired--and preferably in the range 5 to 25 KDa;especially in the range 15 to 20 KDa. Commonly, products of molecularweight about 17 KDa are convenient and accessible.

The active agents may be administered by various modes, conventional inthe art, and the choice depends upon what may be considered mostappropriate for the patient's condition. Thus they may be introduceddirectly into the site of an identified or suspected neuro-degeneration,taking appropriate care that the administration does not itself causeundue damage to the tissue or affect the condition adversely. This maybe done by injection, e.g. central injection, (for example stereotaxicinjection) via hypodermic needles, cannulae, or the like. Forintra-cranial administration pump-assisted apparatus may be used.

Alternatively, they may be administered by indirect methods, so thatthey then migrate within the body from the site of introduction to thesite at which they are required and are to have effect. Thus,administration by infusion can be used, and this can be preferred whendirect access to the site of action is either difficult or considered tobe less desirable, or even may not be easily determinable. The mode bywhich this migration occurs may vary, and may be for example by transferthrough the blood stream or the cerebro-spinal fluid. Thus, the activeagent may be administered by introduction at a peripheral site, forexample by intravenous infusion.

So, for the treatment of brain conditions, the invention gives the usera considerable choice, as administration may be by direct injection intothe intra-cranial cavity, by infusion into the intra-cranial cavity,conveniently by way of the cerebro-spinal fluid, or by introduction at aperipheral site, for example by intravenous infusion.

Combinations of more than one administration technique may be used ifdesired.

Administration may be achieved by conventional apparatus. Furthermorethe active agent may be administered in conjunction with other knowntreatment agents and/or procedures.

The formulations used may be any in which the defined compound (activeagent) is contained in a medium which is safe and compatible with thetissues into which it is to be introduced. Thus the compound may bedispersed or dissolved in a pharmaceutically acceptable carrier (solventor diluent). This is most conveniently water or an aqueousmedium--especially normal saline (an isotonic solution of sodiumchloride in water)--though other media may be used if desired providedthey are pharmaceutically acceptable and compatible with the area to betreated. Thus, conventional adjuvants and additives may be used, forexample in normal saline.

The amount and concentration of the active agent appropriate foradministration may be varied according to the particular need of thepatient and the type and/or severity of the condition to be treated. Theamount of the active agent is most suitably (for injection into the siteof damage) in the range 100 to 10,000 micrograms (and preferably in therange 1000 to 5000 micrograms), on the basis of a patient of 80 kg bodyweight, and an amount of about 2400 micrograms is usually typical andconvenient, though larger or smaller amounts may be used if desired.

If it is considered more convenient, these amounts can be converted into"micrograms per kilogram of body weight" figures by simple calculation,and expressed in this way so that the dosages can be calculated morereadily for various patients.

These amounts are those which it is intended should be at the site atwhich the agent is to act. Thus, if the agent is not introduced directlyinto the desired site, the amounts required for indirect introduction(e.g. by infusion from a peripheral site) should be adjusted so as togive the amount stated above at the site of action. In such cases, theamount used at the peripheral site will usually need to be greater thanin the ranges stated above, but the optimum amount in any individualcase may be determined by clinical factors, having regard to factors asthe patient's condition and the response to the treatment. For example,a relatively high dosage may be most appropriate for a condition ofacute trauma or at the commencement of treatment, while a lower orreduced dosage may be most appropriate for a chronic or continuingcondition calling for an extended period of treatment.

The mode of treatment may be varied to suit the condition being treated.Thus, for example, a single adminstration may suffice in some cases toprovide the desired protection rapidly in acute conditions, but this maybe enhanced by continuing administration, while chronic conditions mayneed continuous administration. The optimum mode and dosage for anyparticular patient or condition can thus be determined by simple trial,and can be modified as treatment continues, in the light of the resultsshown by the patient's response and needs.

The treatment of the present invention may be applied to a variety ofacute and chronic conditions.

Our invention may be applicable to the treatment of relatively long-termneuro-degeneration of non-ischaemic origin (e.g. epilepsy, Alzheimersdisease, Huntingdon's chorea, Downs syndrome, Multiple Sclerosis andParkinson's disease) and neurological damage resulting from chronicinfection for example HIV producing the syndrome of AIDS.

It may also be used for the treatment of ischaemic conditions, forexample cerebral ischaemia (stroke, haemorrhage or brain injury as aresult of trauma) which involve various forms of brain damage and maylead to acute or delayed damage to the brain neurons, and todegeneration--for example after head trauma.

The time of treatment is also significant and can be important.Administration may be before or after an ischaemic condition hasoccurred or is suspected. Administration before an ischaemic conditioncan be of value for prophylactic treatment, for example when the patientor subject is considered to be at risk of an ischaemic condition. Suchconditions could be for example be in cardiac bypass surgery, in which asignificant proportion of patients can suffer minor cerebral damage, orin childbirth, in which the foetus may be liable to problems in thefoetal circulation potentially leading to anoxia and cerebral palsy andthe like. The more common time of administration is after ischaemicdamage has occurred or is suspected, for example in the conditions oftreating a stroke or a head injury, and in such cases it is desirable tomake the administration as soon as possible after the event to get bestresults--preferably within an hour or less, though administration laterthan that time may still be beneficial.

The efficacy of our invention is illustrated by the ability of treatmentby administration of active agents we now specify herein to reducelesions caused by cerebral ischaemia or excitotoxins by up to 70% oreven more, and to reduce the amounts of beta-amyloid precursor protein(beta-APP) by 20% or more which are reduced by cerebral ischaemia.

The invention is illustrated but not limited by the following Examplesand drawings.

EXAMPLE 1

Rats were treated to induce in them a stroke condition (a middlecerebral artery occlusion) by electro-cautery of the middle cerebralartery in the manner customary for such experimental study.

Male Sprague-Dawley rats (Charles River, U.K.) weighing 200-250 g wereused in all experiments. The animals were injected icv (via previouslyimplanted indwelling guide cannulae in the third ventricle of thebrain), 10 minutes prior to surgery, with IL-1 ra (10 micrograms in 4microlitres, i.e. 0.6 nmol, n=12, Synergen, Colo., USA) or 0.9% saline(4 microlitres, control, n=14 ), 30 minutes before and 10 minutes aftersurgery. Cerebral ischaemia was induced by permanent occlusion of theleft middle cerebral artery (R. H. Lye et al., Neurosci.Meth. 22, 133,1987) under halothane anaesthesia (2% in oxygen/nitrous oxide). Allanimals recovered consciousness within 10 minutes after completion ofsurgery and were allowed free access to food and water thereafter. Thedegree of damage, or protection against damage, was assessed byhistological examination of the lesion size to assess the amount ofnon-viable tissue present. The data were summed from multipleexperiments and derived from study of the rat brains 24 hours after thestroke condition commenced and also compared with control animals inwhich the treatment was not applied. The area of damage (mm²) wasassessed by tetrazolium staining on 500μ coronal sections of brain (areacomputed by Seescan image analysis).

The data summarised in FIG. 1 demonstrate that injection of thisantagonist into the third ventricle of the brain 30 minutes before and10 minutes after unilateral focal cerebral ischaemia (MCAo) in the ratinhibits neuronal damage (volume of infarct, measured 24 hours later) byapproximately 50%.

In vehicle-treated ischaemic rats, histological damage (absence ofmitochondrial respiratory activity) occurred reproducibly in basalganglia and neocortex (FIG. 1). Injection of IL-1ra inhibited the extendof damage in these areas, reducing the total volume of the lesion from84±12 mm³ to 42±8 mm³.

Our results therefore show that focal cerebral ischaemia is markedlyinhibited (ca 50%) by cerebral injection of recombinant interleukin-1receptor antagonist protein in the rat.

We have also shown effects of peripheral injection of recombinantinterleukin-1 receptor antagonist protein ("IL-1 ra") on brain damage.

This was carried out by intravenous injection.

The IL-1 ra was given as a dose of 0.5 mg/kg., as a solution in normalsaline.

This was administered as a first injection 30 minutes BEFORE the stroke,followed by a second injection 10 minutes AFTER the stroke.

    ______________________________________                                                         lesion size.                                                 ______________________________________                                        (1) after the above treatment                                                                     57 ± 15 mm.sup.3                                       (2) control (no IL-1 ra)                                                                         104 ± 14 mm.sup.3                                       ______________________________________                                    

This may be expressed as being a 45% reduction in the size of the lesionor as a 45% protection against the effects of the stroke--a greatclinical improvement.

EXAMPLE 2

Study of neuronal death resulting from focal cerebral ischaemia orexcitotoxic damage due to striatal infusion of an NMDA-receptor agonist.

Excitatory amino acids are potent endogenous neurotoxic agents which caninduce neuronal damage and have been proposed as mediators of neuronaldeath following ischaemia, mechanical brain injury, seizures orneuro-degenerative conditions such as Parkinson's disease, Huntingdon'schorea and damage caused by infections such as HIV. The NMDA receptorhas been strongly implicated in these excitotoxic actions of aminoacids. Synthetic antagonists of NMDA receptors (e.g. MK801) are potentneuroprotective agents in ischaemia, while pharmacological NMDA receptoractivation results in neuronal damage. Infusion ofcis-2,4-methanoglutamate, a potent and selective NMDA agonist into thestriatum of rats causes dose-dependent lesions which are markedlyinhibited by pre-treatment with MK801. Data presented in FIG. 2 showthat infusion of 10 nmoles of cis-2,4-methanoglutamate causedreproducible lesions (12.3±1.3 mm³, n=10). Infusion of the interleukin-1receptor antagonist with the NMDA agonist significantly reduced thevolume of lesions induced by the cis-2,4-methanoglutamate by 7.11±4.2%.This indicates that protection against excitotoxic damage is offered bythe IL-1ra agonist.

We have also shown that brain damage caused by striatal infusion ofquinolinic acid in rats is inhibited by IL-1ra. Quinolinic acid causedlesions (assessed from mitochondrial viability) of 27.8±2.4 m³,coinfusion of IL-1ra (10 μg) reduced the size of the lesion to 15.1±2.7mm³ (i.e. by 46%). Quinolinic acid is a naturally occurring moleculewhich, when released in high quantities is toxic to neurons. Excessrelease of quinolinic acid has been reported in the brain in variousneurological conditions (e.g. Huntingdon's chorea and AIDS) and may be acause of neurological damage in these conditions. Therefore, inhibitionof its actions by IL-1ra could be of benefit.

We have thus shown also that the IL-1ra fragment inhibits brain damageinduced by administration of excitotoxic agents (NMDA receptor agonistsor quinolinic acid) to rat brain. Since this mechanism of damageunderlies many other neurological conditions (such as epilepticdegeneration, Huntingdon's chorea, Parkinson's disease and brain damageresulting from infections such as HIV or traumatic brain injury) as wellas ischaemic damage, the IL-ra may be of benefit in each of theseconditions.

EXAMPLE 3 Methods

Rats (Sprague-Dawley 200-250 gms) were anaesthetized with halothane (2%in oxygen/nitrous oxide) and treated so as to induce a neurologicallesion (an infarct). This was achieved by permanent occlusion of theleft middle cerebral artery following an established procedure (Lye R Het al 1987 Neurosci. Meth. 22,133). The animals had previously beenfitted with an indwelling guide cannula in the third ventricle in thebrain and were injected with a solution of IL1-RA (10 micrograms in 4microlitres i.e. 0.6 nmol) or 0.9% saline (4 microlitres) at intervalsof 10 minutes and 30 minutes following the lesion procedure.

A total of 32 animals were used in the various experimental and controlprocedures.

Animals in treated and non-treated groups were examined after 24 hours(n=20 total) and 7 day (n=12 total) survival times.

The brain from each animal was fixed embedded in paraffin and processedfor immunocytochemistry to assess the degree of immunoreactivity to βamyloid precursor protein in the brain (using both polyclonal andmonoclonal antibodies to β amyloid precursor protein).

Results

Lesion size varied from animal to animal. However, increasedimmunoreactivity to the β amyloid precursor protein was consistentlyfound in a region extending some 2 mm from the edge of the infarct.Animals treated with IL1-RA had a reduction in lesion size and alsomanifested a marked reduction of the levels of β amyloid proteinprecursor protein-immunoreactivity in the neurons surrounding thelesion.

Similar results were seen in animals with both 24 hour and 7 daysurvival times.

We claim:
 1. A method of inhibiting or delaying neuronal cell death resulting from over-activation of NMDA receptors in the brain of a human being experiencing a condition of cerebral neurological degeneration arising from or related to cerebral ischaemia, comprising the step ofadministering to said human being in need of such treatment an effective amount of an agent wherein the agent is IL-1 ra.
 2. The method of claim 1 wherein the IL-1 ra is recombinant IL-1 ra.
 3. The method of claim 1 wherein the agent is a fragment of IL-1 ra.
 4. The method of claim 1 wherein the agent is a synthetic IL-1 ra or fragment thereof.
 5. The method of claim 1 wherein the agent is administered such that, at the site of action of the agent, the amount of the agent is between 100 and 10,000 micrograms per 80 kg body weight.
 6. The method of claim 5 wherein the amount of the agent is between 1000 and 5000 micrograms per 80 kg body weight.
 7. The method of claim 6 wherein the amount of the agent is about 2400 micrograms per 80 kg body weight.
 8. The method of claim 1 wherein the agent is administered by injection.
 9. The method of claim 1 wherein the agent is administered at a peripheral site from which it subsequently migrates within the body to the site of the identified or suspected neuro-degeneration.
 10. The method of claim 9 wherein the agent is administered through the blood stream.
 11. The method of claim 9 wherein the agent is administered by intravenous infusion.
 12. The method of claim 1 wherein the agent is administered by transfer through the cerebro-spinal fluid.
 13. The method of claim 1 wherein the agent is administered into the intra-cranial cavity.
 14. The method of claim 1 wherein the agent is administered in a formulation in which it is dispersed or dissolved in a pharmaceutically acceptable carrier.
 15. The method of claim 9 wherein the carrier is an aqueous medium.
 16. The method of claim 15 wherein the aqueous medium is normal saline.
 17. The method of claim 1 wherein the agent is administered continuously.
 18. The method of claim 1 which is applied to a condition of cerebral neurological degeneration selected from the group consisting of a stroke, a haemorrhage and a brain injury resulting from trauma.
 19. The method of claim 1 wherein the agent is administered after ischaemic damage has occurred or is suspected.
 20. The method of claim 1 which is applied to excitotoxic brain damage.
 21. A method of inhibiting or delaying neuronal cell death resulting from over-activation of NMDA receptors in the brain of a non-human mammal experiencing a condition of cerebral neurological degeneration arising from or related to cerebral ischaemia, comprising the step ofadministering to said mammal in need of such treatment an effective amount of an agent wherein the agent is IL-1 ra.
 22. A method of inhibiting or delaying neuronal cell death resulting from over-activation of NMDA receptors in the brain of a human being, the method comprising administering to the human being in need of such treatment a therapeutically effective amount of an agent which inhibits the action of interleukin-1, wherein the active agent is IL-1ra. 